Proper cell function and avoidance of disease requires subcellular targeting and fusion of transport vesicles with acceptor membranes. Vesicle coats, rabs, tethers and SNAREs make up the core of the vesicle trafficking machinery for the endomembrane system. Although the primary point of function of each of these protein families is established, new functional roles at unexpected steps, and new regulatory interactions linking and integrating their functions continue to emerge. In mammals, ER-to-Golgi transport, which represents the rate-limiting step in the secretory pathway and the step most relevant to transport-related diseases, has been extensively characterized in vivo and reconstituted in vitro. In broad terms, ER-to-Golgi transport has been shown to comprise: 1) cargo sorting and vesicle budding mediated by the COPII coat; 2) homotypic COPII vesicle tethering mediated by rab1, p115 and TRAPP1, and fusion mediated by ER/Golgi SNAREs to form pre-Golgi organelles called vesicular tubular clusters (VTCs); and 3) VTC-mediated cargo sorting and transport along microtubules leading to fusion with the Golgi. This project will employ live cell Ca2+ measurements, kinetic assays of ER/Golgi transport in intact mammalian cell lines and in vitro reconstitution of transport phenomena from subcellular fractions to dissect the roles of luminal Ca2+, the Ca2+ sensor ALG-2 and the COPII coat in specific stages of this pathway. The studies are motivated by the broad guiding hypotheses that luminal Ca2+ escaping from pre- Golgi secretory organelles interacts with and regulates the trafficking machinery, significantly impacting cargo transport and/or sorting. The proposed experiments fall under three specific aims: 1) Test the hypothesis that luminal Ca2+ concentrations drop dramatically between the ER and the intermediate compartment. 2) Test the hypothesis that ALG-2 and sec31A transduce the luminal Ca2+ signal to regulate multiple steps in VTC formation and function. 3) Identify Ca2+ targets on COPII vesicles.